Fluorescent nanoparticle composites themselves, process for the preparation of such composites, and use in rapid diagnosis systems with affinity to biological molecules

ABSTRACT

The present invention provides fluorescent nanoparticle composites themselves, the process of preparing such composites, to systems for rapid diagnosis (as “kits”) containing such composites, and to the use of such composites. In a preferential embodiment, the composites of the present invention have an affinity for biological molecules, such as DNA. The present invention also comprises the preparation of probes containing biological material, upon which are added fluorescent nanoparticle composites, making viable a rapid and economic biological diagnosis of, for example, diseases and genetic traits, notably in the medical and veterinarian fields. There is yet the fact that the absorption of radiation in the ultraviolet and visible regions provides advantageous use of its fluorescent properties in photovoltaic or electroluminescent devices, such as organic LEDs, or for the increase in luminous gain of fluorescent lamps, which represents another characteristic of the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of and claims priority tocurrently pending U.S. patent application Ser. No. 12/934,216 filed onMar. 23, 2011, titled “FLUORESCENT NANOPARTICLE COMPOSITES THEMSELVES,PROCESS FOR THE PREPARATION OF SUCH COMPOSITES, AND USE IN RAPIDDIAGNOSIS SYSTEMS WITH AFFINITY TO BIOLOGICAL MOLECULES”, which is a 371application of International patent application Ser. No.PCT/BR2009/000117 filed on Mar. 23, 2009 (Expired), which claimspriority from Brazilian patent application Ser. No. PI0805991-8 filed onMar. 24, 2008. The entire contents of the aforesaid U.S. patentapplication, international application and the aforesaid Brazilianpatent application being incorporated herein by reference.

FIELD OF THE INVENTION

The present invention refers to fluorescent nanoparticle composites.More specifically, it refers to the composites themselves, to theprocess of preparing such composites, to systems for rapid diagnosis (as“kits”) containing such composites, and to the use of such composites.In particular, the composites of the present invention provide, amongother advantages the absorption of radiation in the ultraviolet andvisible regions, with the emission of light in the near ultraviolet andvisible range, including in the colors of deep blue and/or green,providing advantageous use of its fluorescent properties in photovoltaicor electroluminescent devices, such as organic LEDs, or for the increasein luminous gain of fluorescent lamps. Besides that, the composites ofthe present invention have an affinity for biological molecules, such asDNA and RNA, also providing for applications in the medical andveterinarian fields, and in the diagnosis of genetic diseases as well asthose caused by several pathogens.

BACKGROUND OF THE INVENTION Molecular Diagnosis

The molecular diagnosis of diseases and genetic traits is an emergingfield, particularly in the area of clinical analysis. Generally, it usestechniques from molecular biology for the study of DNA/RNA, ofinfectious agents, or of genetic changes in the organism itself, aidingin the diagnosis and prognosis of infectious and genetic diseases.

The more common molecular biology techniques currently used are:enzymatic amplification of the DNA (PCR), digestion of the genomic DNAstrand or of PCR product with restriction enzymes, electrophoreticseparation of the DNA or of the PCR product, hybridization of the DNA orPCR fragments with oligonucleotide probes, DHPLC and cytogeneticmethods. These techniques allow the rapid genotyping of polymorphicmarkers, tracking of uncharacterized mutations. In particular, thecytogenetic methods, based on the microscopic observation of normal andabnormal chromosomes, allow the construction of cytogenetic maps of thegenomes of many species. The FISH (fluorescent in situ hybridization)cytogenetic method is the most direct means of locating molecular andgenetic markers in the cytogenetic map, allowing the integration betweengenetic and molecular markers. Probes are widely used for diagnosis,such as cosmid probes, which are unique sequences connected in smallsegments of certain chromosomes, being useful for the study ofmicrodeletions. Other probes are used to detect translocations andhighly repetitive sequences. However, one should point out that some ofthese techniques still have some limitations, such as false-positivesignals, that can lead to an error in diagnosis.

A well-known molecular diagnosis system is the ELISA (Enzyme-LinkedImmunoSorbent Assay). This immune-enzymatic test allows the detection ofspecific antibodies in the serum of patients, being the first-line testin the diagnosis of HIV (human immunodeficiency virus) infection. Themethod for performing the test is based on the antibody-antigeninteraction, with this test also being capable of detecting othersubstances, such as hormones.

The present invention refers to the fluorescent nanoparticle compositesthemselves, method for the preparation of these composites, system forrapid diagnosis (as “kits”) containing such compounds, and functioningof said “kits”. In particular, the composites of the present inventionhave specific characteristics regarding size and fluorescence, and havean affinity for biological molecules, such as DNA, RNA, and alsoproteins. The method for the preparation of these compounds is alsodescribed in the present invention. Plus, the present inventiondescribes the method of preparation for an adequate probe (named here as“support”) containing biological material of the organism one wishes tostudy. Upon this support the fluorescent nanoparticle composites and thepatient's biological material are added, comprising a diagnostic system,designated here as the ELINOR (from “Enhanced Luminescence fromInorganic/Organic nanocomposites”) test, for the diagnosis of diseasescaused by several pathogens and/or genetic diseases, amongst otherthings. The present invention has application mainly in the medical andveterinarian fields.

The patent literature describes an ample variety of probes for thediagnosis of specific diseases. However, most of the documents deal withmethods that use the PCR molecular biology technique, requiring theamplification of the biological molecule that one wishes to study inorder to perform the diagnosis. One can exemplify the methods for thediagnosis of diseases by the documents presented below.

Document U.S. Pat. No. 6,258,570 deals with a method for the diagnosisof viral meningitis using PCR, as does document U.S. Pat. No. 7,041,255,which uses the same technique to detect infection by the dengue virus.Likewise, the PCR is used for the diagnosis of the human papilloma virus(HPV), as described by document U.S. Pat. No. 6,027,89, and ofStreptococcus pneumoniae, as described by U.S. Pat. No. 6,869,767.

The present invention differs from all of those documents by notrequiring a step of amplification (such as the one performed in the PCRtechnique) in order to perform the molecular diagnosis.

The patent literature also reveals several examples of fluorescentbiosensors containing gold, out of which we highlight the most relevant.

Document US 2007/0059693 describes a biosensor containing a fluorescentsurface, molecules of nucleic acid, and a fluophore. The fluorescentsurface may be a metal, including gold. The molecules of nucleic acidmust have one of the ends bound to the fluorescent surface and the otherend to a fluophore. This molecule of nucleic acid may also have internalhybridization regions that, when hybridized, form a “staple”. In thesecases, the fluophore will be close to the fluorescent surface, allowingfluorescence to occur. The present invention differs from that documentdue to the support surface not being necessarily fluorescent ormetallic, and not requiring that the molecules of nucleic acid form a“staple” in order to emit fluorescence.

Document US 2005/0196876 describes a method for the analysis of thecontent of a biological sample through the contact of the sample with ananoporous biosensor. This biosensor contains probes that bind to thesamples forming complexes that will be bound to a second probe. Thatprobe will be illuminated so as to send a specific fluorescent signal.In an optional configuration, this biosensor may have a layer of gold.The present invention differs from the aforementioned document bydealing with fluorescent nanoparticles containing gold, there being noneed to bind to more than one probe.

Document U.S. Pat. No. 6,773,884 describes a method for the detection ofnucleic acids in which those molecules are put in contact with one ormore nanoparticles of gold bound to oligonucleotides and to fluorescentmolecules. When the hybridization occurs, the interaction of thesemolecules with the oligonucleotides suffers an alteration detectable aschanges in the florescence. The present invention differs from theaforementioned document by dealing with nanoparticles in which the goldis covered by polymers, and by being deposited over the biologicalmolecules studied, there being no need for the presence ofoligonucleotides bound to the nanoparticle.

Document U.S. Pat. No. 7,083,928 describes the detection of negativelycharged polymers using water-soluble cationic polythiophenes. Thenegatively charged polymers include biological molecules such as nucleicacid. This polymer may be bound to a conductive support, such as a goldsurface. When the polymer is detected, there is a change in theelectronic load, fluorescence, or color, The present invention differsfrom the aforementioned document by dealing with nanoparticles of goldcovered by polymers that interact with the biological molecules, withthe gold not being part of the adequate support that will immobilize thebiological molecules.

Therefore, no document was found describing, nor suggesting, thefluorescent nanoparticle composites themselves, their form ofpreparation, the systems containing such composites for use indiagnostic “kits”, or form of functioning for such systems.

SUMMARY OF THE INVENTION

It is one of the objects of the present invention the production offluorescent composites of nanoparticles on themselves, comprising:

-   a) at least one oxidizing agent;-   b) at least one stabilizer agent;-   c) at least one monomer.

Optionally, the oxidizing agent is a salt in which the cation isselected from the group comprising metals chosen from groups 1B to 8B ofthe periodic table.

In a preferential realization, the oxidizing agent is HAuCl₄.

Optionally, the stabilizer agent is a compound comprising a mercaptoand/or silane group.

In a preferential realization, the stabilizer agent is(3-mercaptopropyl)trimethoxy silane (MPS).

In a preferential realization, the monomer is aniline.

In a preferential realization, the fluorescent composites are composedby conducting polymer chains enveloping metallic nanoparticles withlinear sizes of 50 nm or less.

In a preferential realization, the properties of the composites arechanged by variation of their oxidation state and/or change of the pH ofthe medium.

It is an additional object of the present invention, one system forrapid diagnosis comprising:

-   a) at least one fluorescent composite;-   b) at least one short nucleotide sequence;-   c) an appropriate substrate for the immobilization of the referred    sequence;-   d) genetic sample of the patient;

In a preferential realization, the referred sequence of nucleotides ispart of a single stranded DNA.

In a preferential realization, the referred substrate is a glass slide.

In a preferential realization, the referred immobilization is performedby the deposition of approximately 1 .mu.L of a 100 pmol solution ofbiological material on the substrate.

In a preferential realization, the genetic material of the patient isthe “total DNA”, obtained after a simple DNA extraction from a sample ofmaterial provided by the patient.

It is an additional object of the present invention to provide animproved process for diagnosis of genetic and/or infectious diseases. Inan preferential realization, the process of diagnosis of the inventioncomprises:

-   a) to immobilize a short single strand of the nucleotide sequence    that uniquely characterizes the organism to be investigated disposed    on a to least one appropriate support;-   b) to establish physical contact among the immobilized material of    a), the genetic material obtained from the patient and the composite    of fluorescent particles;-   c) to determine the corresponding fluorescence emission signal.

In preferential realization, the genetic material of c) is the “totalDNA”, obtained after a simple extraction of the DNA.

It is yet another object of the present invention to provide the use ofthe fluorescent composites of the invention for the preparation ofphotovoltaic devices, such as solar cells, electroluminescent devices,such as organic LEDs, sensors, or for the increase in the lightingefficiency of fluorescent lamps.

It is yet another object, the present invention provides the use of thecomposites of the invention for the preparation of reagents and/orconsumable items, such as, but not limited to, fluorescent markers, foruse in diagnosis.

In a preferential realization, the intensity of the fluorescenceemission indicates the presence or absence of the biological material ofthe organism to be examined in the genetic sample obtained from thepatient.

In an alternative preferential realization, the composite is combined tomagnetic composites, in such manner to allow the use of externalmagnetic fields to assure the increase and/or separation of thefluorescent fraction that contains the biological material of interest.

In another preferential realization, the composite is prepared by use offunctionalized polymers, i.e., polymers that are bound in a covalentmanner to the short nucleotide sequence, so that the fluorescent taggingof the genetic material (including native and unfolded protein) of thepatient can occur still in solution phase, with no additional need ofits anchorage on a solid substrate.

These and objects of the present invention will be better understood andproperly appreciated after analysis of the detailed description of theinvention and the corresponding accompanying claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic representation of the preparation route to obtainthe fluorescent composites ((Au nanoparticles)/(conducting polymer)).

FIG. 2 is the UV-Visible absorption spectrum of the gold/polyaniline(Au/PANi) composites, where the plasmon band (associated to the reducedgold forming metallic nanoparticles) and the polaron band (associated tothe monomer oxidation process leading to the formation of the polymer).

FIG. 3 is a scanning electron microscope image of the fluorescentcomposites (Au nanoparticles)/(conducting polymer). (Magnifying factorof 4,000×).

FIG. 4 is a scanning electron microscope image of the fluorescentcomposites (Au nanoparticles)/(conducting polymer). (Magnifying factorof 10,000×).

FIG. 5 is a transmission electron microscope image of the fluorescentcomposites (Au nanoparticles)/(conducting polymer).

FIG. 6 is a transmission electron microscope image in dark field of thefluorescent composites (Au nanoparticles)/(conducting polymer). Thelighter regions indicate the presence of metallic nanoaggregatesenveloped by the polymeric chains.

FIG. 7 is a transmission electron microscope image of the fluorescentcomposites (Au nanoparticles)/(conducting polymer) where the metallicnanoaggregates can be seen.

FIG.. 8 is a high-resolution transmission electron microscope image ofthe fluorescent composites (Au nanoparticles)/(conducting polymer).

FIG. 9 is a X-ray diffraction image of the fluorescent composites (Aunanoparticles)/(conducting polymer).

DETAILED DESCRIPTION OF THE INVENTION

The composites of the invention are useful for different applications,including: the preparation of photovoltaic devices, such as solar cells,and electroluminescent devices, as organic LEDs, leading in both casesto a substantial increase in their quantum efficiency; the increase inthe lighting efficiency of fluorescent lamps; the preparation ofreagents and consumable items for diagnosis procedures, amongst otherapplications. The composites of the present invention provide, amongother advantages, the absorption of incident light in the ultraviolet orvisible regions and the emission of light in the ultraviolet and visibleregion, inclusive in the “deep blue” and/or green colors, providing aspecial advantage in their use in photovoltaic devices, such as solarcells, or in electroluminescent devices, as organic LEDs, or for theincrease in the quantum yield of lighting systems, such as fluorescentlamps. In regard to the latter application, the composites of thepresent invention provide an environmentally friendlier and more energyefficient alternative to the phosphors presently used in the internallayer of coverage of fluorescent lamps to assure the ultraviolet quantumcut-off and that are a source of pollution when not properly discarded.The composites of the invention can be prepared so as to provideemission in different colors and with wide-range adjusting intensities,according to the tuning of their composition and preparation manner.

The composites of the present invention have affinity for biologicalmolecules, such as DNA, RNA, or proteins, providing also applications inthe areas of human and animal health and in the diagnosis tests fordiseases caused by different pathogenic agents. In this regard, thefollowing examples do not have the purpose of limiting the range of theinvention, but rather only illustrate one of the innumerable manners ofrealizing the invention.

It is understood by “biological material” the group of compounds thatcomprises, but it is not limited to, DNAs, RNAs, proteins, lipids,peptides, non-codifying RNAs, and/or any other biological material thatcould be represented by a single chain or single strand.

It is understood by “genetic material of the patient” the group ofbiological material that comprises, but it is not limited to, thebiological material of any organism that could be present in a smallamount of blood or obtained from a simple collection of epithelial ormucosa cells, and/or from secretions and/or excretions of the patient.It is understood by “oxidizing agent” is a salt in which the cation isselected from the group comprising metals chosen from groups 1B to 8B ofthe periodic table. This group of compounds comprises, but it is notlimited to, to gold compounds, such as HAuCl₄. Preferentially, the goldatom is in the 3+ oxidation state. However, other salts of metals of the1B to 8B families can be used, provided that their oxi-reductionpotential allows the oxidation of the monomer, leading to the formationof the polymer. The present inventors have prepared other compounds notonly based on Au, but also on Ag and Cu, and using other monomersbesides pyrrole, such as derivatives of aniline and thiophene. In asimilar way, the experts in the field will understand that metals suchas nickel, platinum and palladium can also be used. The presentinventors have also prepared other composites in which the conductingpolymer was used in the presence of metallic oxides, in such manner asto obtain composites that exhibit at the same time properties offluorescence and magnetism. It is understood by metallic oxides,compounds, the general class of compounds containing oxygen and metals,such as, but not limited to, iron and titanium. It is understood by“monomer” any compound that can be polymerized by the oxidizing agent.Namely, it is chosen from the group that comprises, but it is notlimited to, the smallest repetitive unit of a polymer, as those derivedfrom aniline (C₆H₅NH₂), thiophene (C₄H₄S), pyrrole (C₄H₅N), or precursormolecules of the respective polymers, polyaniline, PEDOT((poly(3,4-ethylenedioxythiphene)poly (styrenesulfonate)), PTAA(polythiophene acetic acid) and polypyrrole, and/or a mixture of these.

It is understood by “stabilizing agent”, the group of compounds thatcomprises, but it is not limited to, silanes, such as (3-mercaptopropyl)trimethoxy silane (MPS), (3-mercaptopropyl) methyldimethoxysilane,(3-mercaptopropyl) triethoxysilane e (3-mercaptoethyl)trimethoxysilaneand/or a mixture of them.

It is understood by “alcohol” the group that comprises, but it is notlimited to methanol, ethanol, propanol, butanol, glycerol, ethyleneglucol and/or a mixture of them.

EXAMPLE 1 Synthesis and Characterization of the Nanoparticles Example1.1 Preparation of the Nanoparticles

The preparation of nanoparticles was performed (see scheme in FIG. 1) ina round bottom glass flask containing ethanol (20 mL) and the compounds:aniline (Ani-C₆H₅NH₂) (0.030 mol/L), 3-mercaptopropyl-trimethoxy-silane(MPS-C₆H₁₆O₆SSi) (6.46×10⁻² mol/L) and HAuCl₄.xH2O (0.81 mmol/L), whichwere subsequently added and subject to energetic agitation (1,100 rpm).Aniline (Ani-C₆H₅NH₂) was acquired from VETEC (Brazil) and only usedafter distillation in a Kugelrohr apparatus. The other compounds werebought from Aldrich Co. (USA), and had at least 99% degree of purity.All subsequent experiments were performed in the 48 hours' time intervalafter the mixtures.

Example 1.2 Characterization of the Nanoparticles

Photoluminescence properties were measured by use of a quartz cuvette (1cm and 5 mL) in a PC1 (ISS, USA) spectrofluorimeter at (20±1) ° C. Thesamples were monitored at different pH values by use of two luminescencematrices: (1) in the 200 to 360 nm excitation range and emission in the370 to 600 nm interval; and (2) in the 270 to 330 nm excitation rangeand emission in the 280 to 600 nm interval. Morphological analyses wereperformed by scanning electron microscopy (SEM), by use of a JSM-5900(JEOL, Japan) electron microscope. The samples were placed atop a glasssubstrate and fixed by a carbon tape. After this, the samples werecovered by a thin gold layer by use of a sputtering (BalTec SCD 050).The size of the particles was determined by a light-scattering method byuse of a Zetasizer Nano-ZS90 instrument (Malvern).

Example 2 Characteristics of the Nanoparticles

Gold nanoparticles with diameters of the order of ¹⁸ 5 nm exhibit intheir absorption spectrum a surface plasmon (SP) band centred in 525 nm.The UV-Vis spectrum of the composites is shown in FIG. 2, where once canobserve the strong presence of a SP band at 560 nm. It is known that thewavelength and the intensity of the SP band vary according to the size,shape and the “interparticle” dielectric medium, and that it is alsosensitive to the relative molar fraction (stabilizing agent)/Au [J. Am.Chem. Soc. 2003, 125, 9906]. It is also known that polyaniline (PANi)exhibit two characteristic absorption bands (324 nm and 625 nm) in theUV-Vis region.

In the method used in the present invention, the gold containingcompound (HAuCl₄.xH₂O) acts as an oxidizing agent, i.e., the trigger ofthe aniline polymerization, while a mercapthosilane is included as aco-stabilizer of the formed metallic nanoparticles. In the fluorescencematrix of the polyaniline-gold (PANi-Au) sample, one can verify that thecomposite exhibit luminescent properties in the visible region, sincethe composite presents a peak of photoluminescence centred close to 400nm when excited in the ultraviolet (350 nm) region. The use of goldnanoparticles and conducting polymers in light emitting diodes, whiletrying to increase the electroluminescence stability and quantum yield,was discussed in a recent paper [Chem. Mater.: 2004, 16, 688-692], whereit is proposed that the reason for the observed effects are theincreased roughness of the metallic cathode surface and the improvementof the balance of the injected charges promoted by the metallicnanoparticles. On the other hand, examples of water soluble and highlyluminescent nanoparticles were recently published [Physical ReviewLetters vol. 93(7) 2004, pp. 77402-1 77402-4], where the intenseluminescence was attributed to the formation of metallic aggregates thatwould lead to the injection and transport of charge through the discretelevels of energy. Differently from the above related examples, in thepresent case the method used has allowed the inventors to prepare goldnanoparticles with sizes of the order of 5 nm (or less), enveloped by a“shell” of conducting polymers, whose dielectric properties can bechanged by varying either their oxidation state and/or the pH of themedium where they are dispersed. In this manner, at least in principleone can tune the emission wavelength of the composite by properlyadjusting the dielectric properties of the medium. Measurements of thequantum yield of the first samples of the composites have indicatedvalues in the 1.5 to 7.5% interval; however, modifications in the methodof preparation already implemented have allowed the inventors toincrease the quantum yield, as well as emission of the same system indifferent wavelengths.

Scanning electron microscopy (SEM) shows that the nanoparticles tend toalign themselves in more complex structures (FIGURES. 3 and 4).

Transmission electron microscopy (TEM) images in bright field wereobtained for the composites object of the present invention (FIG. 5),where one can identify the presence of agglomerates with an averagediameter of 50 nm. In addition, in the dark field mode, one can clearlysee a regular and homogeneous distribution of gold nanoparticles (FIG.6). It is important to stress that in light scattering experiments theaverage size of the agglomerates of the composites was estimated to bein the 150 to 300 nm range.

FIG. 7 reveals that there is a monodisperse distribution ofnanoparticles of sizes varying from 2 to 5 nm, even though in some casesformation of geminal particles—a well-known characteristic of goldnanoparticles—could be identified. A High Resolution TransmissionElectron Microscopy (HRTEM) image of the hybrid gold/(conductingpolymer) nanocomposite reveals the presence of crystalline structures(FIG. 8), an observation that is confirmed by examining thecorresponding X-ray diffraction (XRD) pattern (FIG. 9).

Example 3 Diagnosis Kits Containing the Fluorescent NanostructuredComposites

Due to the fact that it is possible to adapt the methodology proposed inthe present invention to large scale production with low capitalinvestment and at a very price per unit, the associated technology hasits low cost and speed of implementation as principal comparativeadvantages over the methods usually adopted in the diagnosis ofinfectious diseases caused by bacteria or virus, factors thataccompanied by a greater generality and flexibility of application. Onecan identify some important characteristics of the use of thefluorescent nanocomposites in diagnosis kits:

-   (1) the specifity towards the presence of a given pathogenic agent    is determined by the nature of the fragment of the biological    material (such as a DNA single strand) immobilized in the probe, so    that the technique do not is limited on that regard, and can be used    for the identification of any organism for which a specific short    sequence of biological material, such as DNA, can be obtained;-   (2) the technology is of general use for the diagnosis of any    disease: whose origin can be: a) attributed to a known pathogenic    agent, or b) associated to the presence of a specific sequence of    biological material (such as DNA or RNA), even if human (and so it    opens the possibility of using the technology for the investigation    not only of diseases already installed but also for the analysis of    genetic tendency of patients with regard to the future development    of hereditary pathologies;-   (3) the amount of biological material to be used in the diagnosis    assays is extremely small (e.g., a volume of 1 μL of a 100 pmol    solution of biological material, such as DNA);-   (4) the preparation of the probes containing the sequence of the    biological materials (such as DNA) is a step that can be adapted to    large scale production, once again at a very low cost;-   (5) the manipulation of the genetic material obtained from the    patient to use in the proposed diagnosis procedure do not require    steps related to separation and amplification of the DNA of    interest, via polymerase chain reaction (PCR) and similar    techniques;-   (6) the result of the diagnosis assay has a conclusive character    (i.e., positive/negative) and it can be obtained in a matter of    minutes, with no need of using any kind of culture medium;-   (7) the result of the diagnosis assay is based in the observation of    the intensity of the fluorescence signal, indicating the presence or    absence of the nucleotide sequence of interest;-   (8) in the case of existence of genetic variation of the pathogenic    agent in different subtypes (as in the case of the dengue virus, for    example), the assay probe can be prepared in such manner as to    contain biological material of each subtype to be investigated, and    hence a single test can provide a conclusive answer with regard to    the presence of any variety of the pathogen in the genetic sample    provided by the patient;-   (9) in the case in which the symptoms exhibited by the patient can    be attributed to a limited number of possible pathogenic agents (as,    for example, in the case of hospital acquired infections, or in the    case of victims of accidents with deep perforations and wounds), the    probe can be prepared in such manner as to contain biological    materials (nucleotide sequence) of each one of the agents, so that    in a single and rapid exam the diagnosis can be conclusive for the    presence of any of them;

Since this technology can be applied to the diagnosis of the presence ofany pathogenic agent, one can choose the nature of the microorganism tobe investigated in appropriated tests, defined from the problems ofpossible interest for the public health of a given country or region.The rapid diagnosis kit here proposed can be used, but is not limited,to the diagnosis of: dengue virus: ii) tuberculosis; iii) hepatitis C;iv) human papillomavirus (HPV), v) leishmaniasis, vi) rapididentification (from within a pre-selected range of options) of thecause of hospital acquired infections; vii) rapid identification ofmeningococcus infections; viii) bioterrorism hazards, besides ix)genetic screening of hereditary diseases (such as Tay-Sachs,phenylketonuria, breast cancer, among others). A few examples arediscussed below.

Example 3.1 Diagnosis of the Presence of the Human Papillomavirus, HPV

The diagnosis procedure uses a short sequence of a single nucleotidestrand consisting of 20 bases of the variety 16 of HPV. The quality ofthe response can be attested when a negative answer was obtainedwhenever the probe was exposed to a double strand of the variety 18 ofHPV with circa of 500 base pairs and a positive answer only when theprobe was exposed to double strand with 500 bases pairs of the variety16 of HPV.

Example 3.2 Diagnosis of the Presence of the Dengue Virus

The diagnosis procedure uses a short single strand consisting of 22bases of the subtype 2 of the dengue virus. The quality of the responseis associated to a negative answer when the probe was exposed to adouble strand non-complementary to the original sequence used and to apositive diagnosis when the probe was exposed to a double strandcontaining 22 base pairs of the subtype 2 of the virus dengue.

Example 3.3 Diagnosis of the Presence of the Human papillomavirus (HPV)and the Sensitiveness of the Response to the Presence of Alleles

The diagnosis procedure uses short single strand sequences of 19(MBL54mt) and 22 (MBL57mt) bases corresponding to human lectin responsesto different HPV varieties, some of them containing mutations inspecific positions that could block the hybridization of the DNA chainsof the pathogenic agent present in the material of the patient. The typeof response (positive or negative answer) obtained, respectively, forhomozygous and heterozygous patients define the sensitiveness of thetechnology as excellent.

In all of the examples above referred, a short sequence of a singlestrand of nucleotide chain (DNA or RNA) was anchored atop a previouslysilanized glass substrate, and afterwards a small drop of the mixture(composite (metal nanoparticle)/(conducting polymer)+(total DNA of thepatient)) was added. The system was subsequently washed with runningdistilled water and, after waiting for about three minutes for drying,the substrate was placed in a fluorescence microscope for analysis. Incase of existence of genetic material of the pathogenic agent in thebiological material obtained from the patient (the “total DNA”), a longnucleotide strand of the pathogenic agent will hybridize to theimmobilized short sequence, and retain a larger amount of fluorescentcomposite: a “positive” answer will then arise. If the hybridization didnot occur, only a smaller amount of the composite Will remain attachedto the short immobilized sequence of nucleotide, and as a consequencethe fluorescence signal will be minimum (basal): the “negative” answer.It has to be noted that in a series of tests with the HPV, one of the 20bases was deliberately altered, changing an original “positive” answerto “negative”; hence, the sensitiveness of the here proposed procedureis able to discriminate the change of a single base in 20.

Yet other applications of the composites object of the present inventioncan be immediately apprehended by the experts in the field, once theyhave been exposed to the present information. Among others, one can callattention to the rapid in situ diagnosis in situations such as:diagnosis of diseases in the battlefield; rapid identification ofanthrax and other forms of bioterrorism contamination; biologicalcontamination of food and beverage products in general, as in the caseof control of quality of grains and cereals; biological assays in thefield for in situ identification and comparative analysis of specimenswith regard to pre-selected biological characteristics (screening in thefield or biobarcoding), eliminating the need of collecting andtransporting redundant material; and methods of forensic identification.In regard to the last subject, the composites of the present inventioncan act as “nanoluminol”; a fairly recent publication of the Universityof San Diego shows that DNA portable detectors may offer substantialadvantages over the present technology. Even tough the technologyadopted in such reference is much more complex and expensive(ion-selective field-effect transistor—ISFET) than that discussed in thepresent invention, it is an important example of the actual need of newdevelopments this area of expertise.

The skilled in the art will immediately recognize the value of thepresent teachings and they also will understand that variations in theforms of executing the invention herein exemplified must be consideredas within the spirit of the present invention and in the general scopeof the accompanying claims.

1-26. (canceled)
 27. Fluorescent nanoparticle composites themselvescharacterized by comprising: a) at least one oxidizing agent; b) atleast one stabilizer agent; and c) at least one monomer.
 28. Thecomposites, according to claim 27, characterized by the fact that saidoxidizing agent is a salt in which the cation is selected from the groupcomprising metals chosen from groups 1B to 8B of the periodic table. 29.The composites, according to claim 27, characterized by the fact thatsaid oxidizing agent is HAuCl₄.
 30. The composites, according to claim27, characterized m that the stabilizer agent is a compound comprising amercapto and/or silane group.
 31. The composites, according to claim 27,characterized in that the stabilizer is3-mercaptopropyl-trimethoxy-silane.
 32. The composites, according toclaim 27, characterized in that the monomer is aniline.
 33. Thecomposites, according to claim 27, characterized in that said compositescomprise polymeric chains with metallic nanoparticles having about 15 nmor less, used alone or together with magnetic metals or metallic oxidesnanoparticles having about 50 nm or less. 34-41. (canceled)
 42. A rapidbiological diagnosis kit characterized by the fact that it comprises: a)at least one fluorescent composite; b) at least one short nucleotidesequence; c) an appropriate substrate or the immobilization of thereferred sequence; d) genetic sample of the patient;
 43. The kit,according to claim 42, characterized by the fact that said nucleotidesequence is RNA or single-stranded DNA.
 44. The kit, according to claim42, characterized by the fact that said substrate is a glass slide,paper and/or polymer strip.
 45. A process for fast biological diagnosischaracterized by comprising: a) immobilizing a short single strand ofthe nucleotide sequence that uniquely characterizes the organism to beinvestigated disposed on at least one appropriate support; b)establishing physical contact among the immobilized material of a), thegenetic material obtained from the patient and the composite offluorescent particles; c) determining the corresponding fluorescenceemission signal.
 46. A method for preparation of diagnostic reactivematerials, comprising: producing a fluorescent composite by combining atleast one oxidizing agent, at least one stabilizer agent and at leastone monomer.
 47. A method for preparation of fluorescent polymers,comprising: producing a fluorescent composite by combining at least oneoxidizing agent, at least one stabilizer agent and at least one monomer.48. A method for preparation of fluorescent polymers that contribute tothe efficiency increase of photovoltaic or electroluminescent devicesthrough a process of energy transfer between the nanocomposites andactive material of the devices, comprising: producing a fluorescentcomposite by combining at least one oxidizing agent, at least onestabilizer agent and at least one monomer.
 49. The method, according toclaim 48, characterized by the fact that the device is a solar cell oran organic LED.
 50. The method, according to claim 48, characterized bythe fact that the device is a fluorescent lamp or illumination system.51. A method for preparation of a fluorescent test for rapid detectionof biological weapons or bioterrorism attacks, comprising: producing afluorescent composite by combining at least one oxidizing agent, atleast one stabilizer agent and at least one monomer.